Healthy animal cells require, among other conditions and materials, the movement of various inorganic ions across the cell membrane to be maintained such that a proper balance of the ions provide the requisite electrical potential across the cellular membrane as well as a life-promoting internal ionic strength. For example, Na.sup.+, Cl.sup.-, K.sup.+, and Ca.sup.++ are known to cross cell membranes in animals such that K.sup.+ and Ca.sup.++ are accumulated intracellularly to a varying extent in different cells at different times of development, whereas Na.sup.+, in large measure, is excluded from the interior of a cell. The cross-cellular movement of these ions is mediated by Na.sup.+ /K.sup.+ - and Ca.sup.++ -dependent ATPases that are membrane bound at the sites of appropriate ion channels. Chloride ion was believed to permeate animal cells by passive means to equilibrate in concentration between the external and internal fluid, resulting in an underrepresentation of Cl.sup.- intracellularly in consequence of the overall negative intracellular change. Conductance of chloride, however, has been shown to be mediated actively as well, by means of a Cl.sup.- channel (see Edwards, Neuroscience, 7, 1335-1366 (1982)).
Results from research directed to the pathology of cystic fibrosis ("CF") has provided information on the ill-effects that an ion conductance impairment at the cellular level can have on a person's health, at many levels. CF was known to have a genetic basis because of its differential incidence among white Americans (between 1/1600 and 1/2000 live births) as compared to African Americans (about 1/17,000 live births). Indeed, research over the past decade has revealed that a heritable discrete gene mutation is associated with the clinical symptoms of CF, including abnormal exocrine gland and lung functions.
More specifically, CF is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, the most common of which is the deletion of a phenylalanine residue at position 508 (Phe.sup.508 ; see FIG. 1 for a physical map of the CFTR protein). The mutated CFTR protein is referred to as .increment.F508, the site of which is within the first nucleotide binding fold (NBF-1). Schoumacher et al., Proc. Natl. Acad. Sci., 87, 4012-4016 (1990); Riordan et al., Science, 245, 1066-1073 (1979). More specifically, this mutation is located in a lengthy internal segment of the NBF-1, flanked on the N-terminal side by the Walker A sequence (amino acid position 458-471; also referred to as "CFTR[458-471]") and on the C-terminal side by the contiguous C domain (amino acid position 548-560; also referred to as "CFTR[548-560]") and the Walker B domain (amino acid position 561-573; also referred to as "CFTR[561-573]"). The physiological role of the intervening sequence located between CFTR[471] and CFTR[561], which includes the polypeptide sequences of I.alpha., I.beta., and Io (see FIG. 1), is unknown.
Certain mutations in the CFTR gene, such as that resulting in .increment.F508, cause an abnormal potential difference across CF epithelia. The abnormality is due to a reduced cellular apical chloride (Cl.sup.-) conductance. Consequently, chloride and sodium transport across epithelial membranes of an individual afflicted with CF, for example, is abnormal. It is also known that cells carrying the .increment.F508 mutation have higher than normal CFTR protein bound to their endoplasmic reticulum (hereinafter, "ER"), although wild type cells also retain and degrade a substantial amount of CFTR protein in their ER, albeit much less so than the .increment.F508 mutant. Ward et al., J. Gen. Physiol., 104, 33a (1994).
This CFTR mutation apparently is responsible for pathophysiological changes in the respiratory system, among others. Nearly all patients suffering from the disease develop chronic progressive disease of the respiratory system. In the majority of cases, pancreatic dysfunction occurs, and hepatobiliary and genitourinary diseases are also frequent.
Although survival of cystic fibrosis patients has improved in recent years, the median survival is still only about 28 years despite the development and implementation of intensive supportive and prophylactic treatment. Present efforts to combat the disease have focused on drugs that are capable of either activating the mutant CFTR gene product or otherwise causing additional secretion of Cl.sup.- from affected cells. Gene therapy is another area of active research, wherein the anion conductance deficit may be repaired by the introduction of a recombinant wild-type CFTR gene, i.e., a CFTR gene that lacks a mutation that results in the abnormality.
Encouraging clinical results have been reported recently for the use of aerosols containing either amiloride (Knowles et al., N. Engl. J. Med., 322, 1189-1194 (1990)) or a mixture of ATP and UTP (Knowles et al., N. Engl. J. Med., 325, 533-538 (1991)), which slow the accumulation of Cl.sup.- in the epithelium of the trachea.
Other drugs that purportedly are useful in the treatment of CF have been described. For example, U.S. Pat. No. 4,866,072 describes the use of 9-ethyl-6,9-dihydro-4,6-dioxo-10-propyl-4H-pyrano(3,2-g)quinoline-2,8-dica rboxylic acid or a pharmaceutically acceptable derivative thereof in the treatment of CF. U.S. Pat. No. 4,548,818 describes the use of a 3-alkylxanthine to treat chronic obstructive pulmonary disease (COPD). U.S. Pat. No. 5,032,593 describes the use of a 1,3-alkyl substituted 8-phenylxanthine or a pharmaceutically acceptable salt thereof in the treatment of bronchoconstriction. U.S. Pat. No. 5,096,916 describes the use of an imidazoline .alpha.-adrenergic blocking agent and vasodilator, such as tolazoline, in the treatment of COPD, including cystic fibrosis, chronic bronchitis and emphysema, or COPD in association with asthma.
Historically, theophylline has been administered to asthmatic and CF patients to enhance lung function. Such lung function enhancement is caused principally by bronchodilation, which is due to the action of theophylline on smooth muscles and inflammation. Theophylline has been shown not only to inhibit phosphodiesterase, but also to antagonize adenosine receptors. Accordingly, because theophylline acts at more than one site, it obviously lacks specificity, thus reducing its usefulness to treat CF. In view of the fact that antagonism of the A.sub.1 adenosine receptor, not inhibition of phosphodiesterase, has been shown to result in stimulating chloride efflux from CF cells, such lack of specificity could result in undesired side effects, such as detrimental effects to cardiac, renal, and/or central nervous system tissue. In addition, large doses of theophylline must be administered to achieve a beneficial effect, thus increasing the risk of side effects from the high toxicity of the compound.
Other compounds that resemble theophylline in basic structure have been tested but have not been found to be useful in evoking chloride efflux from CF cells and, therefore, has no or little potential in the treatment of cystic fibrosis. For example, 3-isobutylmethyl xanthine (IBMX), which is structurally similar to theophylline, is nonspecific in activity and highly toxic and, therefore, lacks utility in the treatment of CF. Also ineffective in the activation of chloride efflux are the compounds 2-thio-8-cyclopentyl-1,3-dipropylxanthine (2-thio-CPX), 1,3-dipropyl-8-noradamantylxanthine (KW-3902), and 1,3-dimethyl-8-cyclopentylxanthine (CPT) (see U.S. Pat. 5,366,977). Similarly, substitution of the propyl group at position 1 or 3 of CPX (1,3-dipropyl-8-cyclopentylxanthine) with a one-carbon group generates a compound that is ineffective in activating chloride efflux from CF cells. Clearly, minor structural differences have a significant, if not substantial, impact on the effectiveness of a given compound in the treatment of CF. Accordingly, it will be particularly useful if a method were available for screening compounds for an ability to promote chloride ion conductance in affected cells, which, in turn, would be a candidate therapeutic agent for treatment of cystic fibrosis patients.
The '977 patent, cited above, and Eidelman et al., Proc. Natl. Acad. Sci. USA, 89, 5562-5566 (1992), disclosed that CPX is a potent A.sub.1 adenosine antagonist that promotes chloride efflux from a human epithelial cell line (CFPAC-1). The CFPAC-1 line, further described in Example 6, expresses the aforementioned CFTR .increment.F508 mutation and can be viewed as a first generation screening material for CF-active compounds. Although CPX and its related xanthine amino congeners disclosed in the '977 patent have been shown to be relatively non-toxic and therefore potentially useful for CF treatment, the fact that such compounds have an antagonistic effect on A.sub.1 -adenosine receptors indicates that such treatment probably will have an additional impact on an animal that is unrelated to the CF affliction. Accordingly, use of such compounds, indeed any compound known to have multiple targets of activity, preferably is avoided because the use of such compounds is, at least, potentially detrimental.
A drug of high potency, low toxicity, and little or no specificity for adenosine receptors, thus, would be a highly desirable and promising therapeutic agent for the treatment of cells having a reduced apical Cl.sup.- conductance, such as cystic fibrosis cells. Such a drug would not only find utility in the treatment of cystic fibrosis rer se but would be therapeutically useful in the treatment of COPD in general.
It is an object of the present invention to provide a method for identifying compounds that can activate an impaired chloride conductance channel. It is also an object of the present invention to provide a method of correcting the reduced Cl.sup.- conductance of cells that are impaired in such conductance. It is another object of the present invention to provide a method of treating cystic fibrosis cells. It is yet another object of the present invention to provide a method of treating cystic fibrosis cells having a deletion involving phenylalanine at amino acid position 508 of the cystic fibrosis transmembrane regulator. It is also an object of the present invention to provide compounds useful in such methods, in particular novel xanthine derivatives that have little or no affinity for the adenosine receptors but have an ability nonetheless to ameliorate the Cl.sup.- imbalance of CF cells by stimulating Cl.sup.- efflux.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.